In recent years, many attempts have been made for stabilizing drugs by chemical modification of physiologically active proteins and enzymes. Among these attempts, chemical modification with a polyoxyalkylene derivative has long been made, and polyoxyalkylene derivatives having carboxyl group at the end of the molecule are known as typical examples of such a polyoxyalkylene derivative.
It is already disclosed that polyoxyalkylene compounds having carboxyl group at the end can generally be transformed into an activated polyoxyalkylene derivative substituted with succinimidyl group by the reaction with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide to increase the reactivity with substances for the modification such as physiologically active proteins (the specifications of Japanese Patent Application Laid-Open No. Showa 62(1987)-185029, Japanese Patent Application Laid-Open No. Showa 63(1988)-60938, and Japanese Patent Application Laid-Open No. Heisei 4(1992)-164098).
Heretofore, for preparation of a polyoxyalkylene derivative substituted with succinimidyl group, a polyoxyalkylene compound having carboxyl group at the end is brought into reaction with N-hydroxysuccinimide in an inert solvent such as dimethylformamide in the presence of dicyclohexylcarbodiimide. After the reaction has been completed, formed dicyclohexylurea is removed by filtration, and the filtrate is added into ethyl ether or petroleum ether dropwise to precipitate the polyoxyalkylene derivative substituted with succinimidyl group as the product (for example, the specification of Japanese Patent Application Laid-Open No. Showa 62(1987)-89630, and A. Abuchowski et al., Cancer Biochem. Biophys. Volume 7, Pages 175 to 186, published in 1984).
In recent years, it has been required that polyoxyalkylene derivatives used for chemical modification of physiologically active proteins or drug delivery systems using liposome be produced in a controlled environment such as a clean room. In the production of polyoxyalkylene derivatives substituted with succinimidyl group, using a large amount of an ether in the crystallization process is not preferable in view of handling because ethyl ether and petroleum ether which have heretofore been used are volatile and there is the possibility of inflammation. When the precipitation is conducted by using petroleum ether, complete removal of dicyclohexylurea formed from dicyclohexylcarbodiimide during the reaction is sometimes difficult. When dicyclohexylurea is left remaining in the polyoxyalkylene derivative substituted with succinimidyl group, an aqueous solution of the derivative becomes turbid, and using such a derivative as a raw material for drugs is not desirable.